A biochemical kinetic model of folate metabolism in breast cancer cells has been formulated and used to investigate the mechanisms of inhibition of purine and pyrimidine synthesis by the drug methotrexate (MTX). The model consists of two nearly independent components, one accounting for the polyglutamation of MTX and the other accounting for various folate interconversions. Normal folates are all assummed to be highly glutamated at about the pentaglutamate level. The MTX polyglutamation component was successfully modelled by a scheme allowing for glutamation, hydrolysis, and efflux of drug polyglutamates. Model parameters for the second component were derived from a large body of MCF-7 cell line folate data, consisting principally of folate pool concentrations measured by HPLC in both drug-treated and drug free cells, and rate constants determined in kinetic studies of isolated enzyme preparations. When applied to drug-free MCF-7 cells, the model was capable of reproducing experimental folate concentrations (dihydrofolate (FH2), 5-methyl-, 10-formyl-, 5,10-methylene-, and unsubstituted